Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Oxidoreduction of cytochrome b in the presence of antimycin

1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators.

2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.

3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.

4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.

5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.

6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c₁ (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH₂/QH^• couple and reduction of cytochrome b.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

The electron transport system of Acetobacter suboxydans with particular reference to cytochrome o

1. The nature and distribution of the electron transport system of Acetobacter suboxydans (ATCC 621) has been investigated, with particular reference to cytochrome o.

2. A highly active membrane-bound electron transport system has been demonstrated, and functional roles suggested for ubiquinone, two c-type cytochromes ( peaks at 549 and 553 nm at — 196°), and two b-type cytochromes ( peaks at 558 and 564 nm at — 196°).

3. Evidence is presented suggesting that both the b-type cytochromes may be terminal oxidases of the cytochrome o type, and that cytochrome o (558) has an O₂ affinity approx. 10 times greater than cytochrome o (565), and a CO affinity only half as great.     

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c₁, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of .

2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.

3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c₁ until a ratio of about 2b (total): 1c₁ (allowing for the cytochrome c₁ present in the cytochrome b preparation) was reached.

4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c₁. It is equal to about one half the amount of native cytochrome b.

5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na₂S₂O₄. Denatured cytochrome b does not quench the fluorescence.

6. Since preparations of cytochrome b active in reconstitution contained cytochrome c₁ in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c₁. The stimulatory action of cytochrome c₁ may be due to the restoration of a damaged membrane conformation.

7. Based on the assumption that the bc₁ segment of the respiratory chain contains 2b:1c₁:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c₁. These are 25.6 and 20.1 mM⁻¹×cm⁻¹ for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.     

Respiratory cytochromes of Euglena gracilis

1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.

2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.

3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.

4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.

Spectral study of b cytochromes in yeast mitochondria and intact cells

Three types of b cytochromes are demonstrated in Candida utilis mitochondria. One of these b cytochromes has a symmetrical -band at 561.5 nm at room temperature. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate both in coupled and uncoupled mitochondria. The second b cytochrome has a double -band at 565 nm and 558 nm. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate in coupled mitochondria, but in uncoupled mitochondria it is slowly reduced after anaerobiosis and this reduction rate is enhanced by antimycin A addition. Thus the oxidation-reduction state of this cytochrome is energy dependent. The first cytochrome is spectroscopically identified as cytochrome b_K and the second as cytochrome b_T. The third b cytochrome has an -band around 563 nm (b₅₆₃) and is reduced slowly after anaerobiosis in uncoupled mitochondria but faster than the b_T. Further properties of this component are not known. Midpoint potentials of cytochromes b_T, b₅₆₃ and b_K are approximately −50 mV, +5 mV, and +65 mV, respectively.

In intact cells, cytochrome b_T is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome b_T while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome b_T.     

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

The redox properties of the cytochromes of purified Complex III

Purified Complex III from beef heart contains two b cytochromes: a high-potential (E_{m 7.2} = +93 mV) cytochrome b-562 which can be enzymatically reduced, and a low-potential (E_{m 7.2} = −34 mV) cytochrome b-565 which is reduced only by dithionite. The two components each contribute approximately 50% to the total cytochrome b of Complex III. Cytochrome c₁ of Complex III titrates with a half-reduction potential of +232 mV.     

Interaction of antimycin with cytochrome b-561: A study in secretory granules and in plasma membrane isolated from chromaffin cells of bovine adrenal medulla

Cytochrome b-561 in chromaffin granules interacts with antimycin and its -peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b561 in secretory granules other than its accredited energy conserving electron transport property is projected.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

Energy transduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the CO-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E′₀ at pH 7.0 +413±5, +270±5, +148±5, +56±5 and −32±5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b₁₄₈, b₅₆, and b₋₃₂) vary as a function of pH with a slope of 30 mV per pH unit.

2. In the presence of a Co/N₂ mixture, the apparent E′₀ of cytochrome b₂₇₀ shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b₁₅₀. The effect of CO on the midpoint potential of cytochrome b₂₇₀ is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain.

3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c₂ and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c₂ in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c₂ nor the CO-binding cytochrome cc′ are involved in this pathway.

4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b₂₇₀, can be classified as a typical cytochrome “o” and considered the alternative CO-sensitive oxidase.     

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes (b_557.5, b₅₆₀, and b_562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c₁, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b_557.5, b₅₆₀, b_562.5, c₁ and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.

2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b₅₆₀, b_562.5 and c₁, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b_557.5 was found in Complex II (succinate-ubiquinone reductase).

3. 3. Cytochrome b₅₆₀ was readily reduced by NADH or succinate, but b_562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c₁ with ascorbate inhibited the subsequent reduction of b_562.5 by substrates. These results indicate that b₅₆₀ and b_562.5 correspond, respectively, to b_K and b_T previously described by Chance et al.¹⁴ (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).

4. 4. Similar to b₅₆₀, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c₁ inhibits its subsequent reduction by substrates. This property is similar to that of b_562.5.

5. 5. Cytochrome b_557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b_557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b_557.5 reoxidation by fumarate.

6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c₁. The spectrum of cytochrome c₁ at 77 °K as modified by 3% (v/v) ethanol is shown.

The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans

Mucidin similar to antimycin inhibits the electron flow to cytochrome c and the enzyme activities dependent on cytochrome c reduction in the cells of Paracoccus denitrificans, but it does not inhibit the electron flow to nitrate reductase and cytochrome o. Unlike antimycin mucidin does not permit a residual electron flow through the cytochrome bc₁ region. In the presence of antimycin the electron flow to nitrate is lower than in using mucidin in contrast with a higher extent of cytochrome b reduction. This result is in contradiction to the participation of the constitutive cytochrome b as an electron donor in the nitrate reduction.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

Rhodobacter capsulatus MT113: A single mutation results in the absence of c-type cytochromes and in the absence of the cytochrome bc1 complex

MT113, a nonphotosynthetic mutant of Rhodobacter capsulatus previously characterized as lacking cytochrome c₂ is shown to lack also cytochrome c₁, the Rieske iron-sulfur cluster and the antimycin sensitive semiquinone Q_c, all components of the cytochrome bc₁ complex. Although MT113 contained b-type cytochromes and other iron-sulfur clusters at nearly wild-type level, it lacks c-type cytochromes. Based on antibody detection, c₂ apoprotein was absent in MT113, however the apoproteins corresponding to the cytochromes b and c₁ and the Rieske iron-sulfur cluster were present in reduced amounts. Genetic analysis indicated that the lesion appears to be due to a single mutation which is not localized in the structural genes of cytochrome c₂ or the bc₁ complex. These data taken together suggest that the pleiotropic mutation in MT113 might be related to the biosynthesis of c-type cytochromes.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.